Literature summary for 2.1.1.235 extracted from

Carney, A.E.; Holden, H.M.
Molecular architecture of TylM1 from Streptomyces fradiae: an N,N-dimethyltransferase involved in the production of dTDP-D-mycaminose (2011), Biochemistry, 50, 780-787.

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Application

Application	Comment	Organism
medicine	the enzyme is involved in biosynthesis of D-mycaminose. D-Mycaminose is an unusual dideoxy sugar found attached to the antibiotic tylosin, a commonly used veterinarian therapeutic	Streptomyces fradiae

Cloned(Commentary)

Cloned (Comment)	Organism
expression in Escherichia coli	Streptomyces fradiae

Crystallization (Commentary)

Crystallization (Comment)	Organism
hanging drop method of vapor diffusion method, high resolution X-ray structures of TylM1, one in which the enzyme contains bound SAM and dTDP-phenol and the second in which the protein is complexed with S-adenosyl-L-homocysteine and dTDP-3-amino-3,6-dideoxyglucose, its natural substrate. Combined, these two structures, solved to 1.35 A and 1.79 A resolution, respectively, show the orientations of SAM and the dTDP-linked sugar substrate within the active site region. Specifically, the C-30 amino group of the hexose is in the correct position for an in-line attack at the reactive methyl group of S-adenosyl-L-methionine. High-resolution X-ray models show that the observed perturbations in the kinetic constants of the mutant enzynes H123A and H123N are not due to major changes in their threedimensional folds. Most likely the proton on the C-3' amino group is transferred to one of the water molecules lining the active site pocket as catalysis proceeds	Streptomyces fradiae

Crystallization (Comment)

Organism

hanging drop method of vapor diffusion method, high resolution X-ray structures of TylM1, one in which the enzyme contains bound SAM and dTDP-phenol and the second in which the protein is complexed with S-adenosyl-L-homocysteine and dTDP-3-amino-3,6-dideoxyglucose, its natural substrate. Combined, these two structures, solved to 1.35 A and 1.79 A resolution, respectively, show the orientations of SAM and the dTDP-linked sugar substrate within the active site region. Specifically, the C-30 amino group of the hexose is in the correct position for an in-line attack at the reactive methyl group of S-adenosyl-L-methionine. High-resolution X-ray models show that the observed perturbations in the kinetic constants of the mutant enzynes H123A and H123N are not due to major changes in their threedimensional folds. Most likely the proton on the C-3' amino group is transferred to one of the water molecules lining the active site pocket as catalysis proceeds

Streptomyces fradiae

Protein Variants

Protein Variants	Comment	Organism
H123A	mutant protein retains catalytic activity, the kcat/Km is reduced to 1.8% compared to the wild-type enzyme	Streptomyces fradiae
H123N	mutant protein retains catalytic activity, the kcat/Km is reduced to 0.37% compared to the wild-type enzyme	Streptomyces fradiae

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.045	-	dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	pH 8.5, 37°C, wild-type enzyme	Streptomyces fradiae
0.67	-	dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	pH 8.5, 37°C, mutant enzyme H123A	Streptomyces fradiae
0.93	-	dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	pH 8.5, 37°C, mutant enzyme H123N	Streptomyces fradiae

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
2 S-adenosyl-L-methionine + dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	Streptomyces fradiae	the enzyme is involved in biosynthesis of D-mycaminose. It is synthesized by the Gram-positive bacterium Streptomyces fradiae as a dTDP-linked sugar	2 S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-3,6-dideoxy-alpha-D-glucopyranose	-	?

Organism

Organism	UniProt	Comment	Textmining
Streptomyces fradiae	P95748	-	-

Purification (Commentary)

Purification (Comment)	Organism
-	Streptomyces fradiae

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
2 S-adenosyl-L-methionine + dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	-	Streptomyces fradiae	2 S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-3,6-dideoxy-alpha-D-glucopyranose	-	?
2 S-adenosyl-L-methionine + dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	the enzyme is involved in biosynthesis of D-mycaminose. It is synthesized by the Gram-positive bacterium Streptomyces fradiae as a dTDP-linked sugar	Streptomyces fradiae	2 S-adenosyl-L-homocysteine + dTDP-3-dimethylamino-3,6-dideoxy-alpha-D-glucopyranose	-	?

Synonyms

Synonyms	Comment	Organism
TylM1	-	Streptomyces fradiae

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
37	-	assay at	Streptomyces fradiae

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.0126	-	dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	pH 8.5, 37°C, mutant enzyme H123N	Streptomyces fradiae
0.046	-	dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	pH 8.5, 37°C, mutant enzyme H123A	Streptomyces fradiae
0.172	-	dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	pH 8.5, 37°C, wild-type enzyme	Streptomyces fradiae

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8.5	-	assay at	Streptomyces fradiae

General Information

General Information	Comment	Organism
physiological function	the enzyme is involved in biosynthesis of D-mycaminose. It is synthesized by the Gram-positive bacterium Streptomyces fradiae as a dTDP-linked sugar	Streptomyces fradiae

kcat/KM [mM/s]

kcat/KM Value [1/mMs^-1]	kcat/KM Value Maximum [1/mMs^-1]	Substrate	Comment	Organism
0.0135	-	dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	pH 8.5, 37°C, mutant enzyme H123N	Streptomyces fradiae
0.069	-	dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	pH 8.5, 37°C, mutant enzyme H123A	Streptomyces fradiae
3.8	-	dTDP-3-amino-3,6-dideoxy-alpha-D-glucopyranose	pH 8.5, 37°C, wild-type enzyme	Streptomyces fradiae